Assessment of allelic variation in vitamin D receptor correlated to bone density or turnover

ABSTRACT

The present invention provides a genetic test for assaying predisposition to and/or resistance to high rates of bone turnover, development of low bone mass and responsiveness or otherwise to therapeutic modalities. This is a specific model for use in prediction of osteoporosis and likely response to preventive or therapeutic modalities. It is a general model of allelic variation in transcriptional regulators determining physiological set-points and thus susceptibility or resistance to certain pathophysiological states.

FIELD OF THE INVENTION

The present invention relates to a method of identifying allelicdifferences in trans-acting factors as a means of identifyingindividuals at risk to suffer from an adverse pathophysiologicalcondition. The method of the present invention is particularly useful inassessing allelic variations in the vitamin D receptor gene and therebypredicting predisposition to low or high bone density. Moreover thesevariants could be used to predict long-term risk of osteoporosis as wellas predicting response to different modalities of therapy. This effectis also a model of determination of predisposition to or resistance toother pathological or physiological variations due to othertranscription factor gene variants and thus determining risk of diseaseand of response to therapy. Such transcriptional regulators could be,but are not limited to, ligand-activated gene regulators. such as thesteroid/retinoid/thyroid hormone receptor gene family.

BACKGROUND TO THE INVENTION

Vitamin D functions as a potent regulator of bone and calciumhomeostasis as well as of cellular differentiation and replication inmany target tissues. It acts as its dihydroxylated metabolite(1,25-dihydroxyvitamin D, or calcitriol) through the highly specificvitamin D receptor (1). This trans-acting transcriptional activatorprotein mediates calcitriol action in the regulation of the expressionof target genes. Cloning the vitamin D receptor gene (2,3) showed it tobe a member of the ligand-activated receptor superfamily that includesthe receptors for steroid hormones (glucocorticoids, progesterone,estrogen, androgen, and mineralocorticoids) as well as thyroid hormonesand vitamin A derivatives (4,5), natural regulators of a large number ofphysiological and developmental processes. The mechanisms by which thesereceptor proteins mediate the regulation of gene expression has been asubject of intense research. Rare overt mutations have been identifiedthat compromise the function of receptors and that cause majorfunctional disorders in humans and animals. For example, mutations inthe vitamin D receptor gene, resulting in vitamin D-resistant rickets(6), and in the androgen receptor, resulting in androgen insensitivity(7), have been reported, and in the estrogen receptor gene an infrequentnatural polymorphism has been correlated with a high rate of spontaneousabortion (8). However, despite a wealth of molecular information, littleis known of the potential contribution of natural allelic variation inreceptor genes to diversity of response to steroidal hormones in normalphysiology and in disease states.

Osteoporosis is a major public health problem among the elderly in mostWestern countries involving both enormous health care costs anddebilitating long-term effects (Riggs NEJM). Since therapy ofestablished osteoporosis remains far from satisfactory, prevention isthe best choice. Preventative strategies for osteoporosis must focusupon development of peak bone density in early adulthood andminimisation of age-related and postmenopausal bone loss. Evidence fromtwin and family studies have shown strong genetic effects on peak bonedensity that is modifiable by hormonal factors, nutrition and life style(Kelly et al, OI). Twin studies have demonstrated that monozygotic twinpairs have a much greater concordance for axial and appendicular bonedensity than do dizygotic pairs. Analysis of these data indicated thatthese genetic factors account for approximately 75% of the totalvariation on bone density. This effect has been confirmed inmother-daughter pair studies. The present inventors analysed thepotential mechanisms of this genetic effect in the twin model. Thepresent inventors found that the genetic effect was apparent in certainbiochemical indices of bone turnover, such as osteocalcin, a marker ofbone formation. Moreover amongst dizygotic twins the higher osteocalcinlevel was associated with the lower bone density. The present inventorshave also found that the genetic effect can be shown with equal strengthin another marker of bone formation, i.e., procollagen type I C-terminalpropeptide and less strongly in a marker of bone breakdown, collagentype I C-terminal telopeptide. Under normal circumstances bone formationand bone breakdown are tightly linked or "coupled" in the twinphysiological process of bone turnover. Thus the somewhat surprisingresults from the twin studies indicate that the bone formation markers,as markers of bone turnover, predict bone density and that geneticregulation of bone turnover is the pathway of the strong genetic effecton bone density.

The cross-sectional data on bone density in twins suggested that asingle gene or set of genes is responsible for the genetic effect onbone density. However, it was unknown how this effect is mediated andwhich gene or genes influence bone density. In recent studies, usingrestriction fragment length polymorphism, the present inventors haveshown common allelic variation in the vitamin D receptor (VDR) locuspredict osteocalcin, independent of age, sex or menopausal status(Morrison et al, PNAS). The vitamin D receptor gene, as the activehormonal form of vitamin D (1,25-dihydroxyvitamin D) is an importantcentral regulator of bone and calcium homeostasis modulating intestinalcalcium absorption, bone formation, recruitment of the bone resorbingcell (osteoclast) and bone resorption per se as well as parathyroidhormone production and vitamin D's own activation in the kidney. Becauseof the likelihood that any alterations in the receptor for the activehormonal form of vitamin D could have such wide effects, the effect ofthese common VDR gene alleles on bone density was examined using a twinmodel. In the twin model, within-pair comparisons eliminate age andvarious cohort effects as confounders.

The studies have shown that common allelic variants in the VDR genepredict differences in bone density and account for 50-75% of the totalgenetic determination of bone density in the spine and hip.

It is believed that this a clear example that genotypic variations intranscriptional regulators of genes encoding regulatory and/orstructural proteins, determine physiological set-points andpredisposition to pathophysiological states with implications forsusceptibility to disease and for determining likely responses totherapy.

Accordingly in a first aspect the present invention consists in a methodof assessing in an individual's predisposition to a pathophysiologicalstate and/or likely response to therapy comprising analysing genotypicvariations in transcriptional regulators of genes encoding regulatoryand/or structural proteins.

In a second aspect the present invention consists in a method ofpredicting predisposition of an individual to low or high bone densitycomprising analysing allelic variation within the vitamin D receptorgene of the individual.

in a preferred embodiment of the present invention the analysiscomprises restriction fragment length polymorphism using endonucleasedigestion.

In a further preferred embodiment of the present invention a segment ofthe vitamin D receptor is amplified using polymerase chain reactionprior to endonuclease digestion.

In yet a further preferred embodiment of the present invention theendonuclease is selected from the group consisting of Bsm1, Apa1, EcoRvand Taq1, and is most preferably Bsm1.

In another preferred embodiment of the present invention the segment ofthe vitamin D receptor is amplified using a pair of primers selectedfrom the group consisting of

5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' (SEQ ID:NO2)

and 5'-AACCAGCGGAAGAGGTCAAGGG-3'λ (SEQ ID:NO3);

and 5'-CAGAGCATGGACAGGGAGCAAG-3' (SEQ ID:NO4)

and 5'-GCAACTCCTCATGGCTGAGGTCTCA-3' λ (SEQ ID:NO5).

In a second aspect the present invention consists in a primer pairderived from the sequence of the VDR gene shown in Table 5 for use inamplifying a segment of the VDR gene using polymerase chain reaction,the segment including at least one of the Bsm1, Apa1 or Taq1 cut sitesas shown in Table 5.

In a preferred embodiment of this aspect of the present invention theprimer pair is

5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' and

5'-AACCAGCGGAAGAGGTCAAGGG-3',or

5'-CAGAGCATGGACAGGGAGCAAG-3' and

5'-GCAACTCCTCATGGCTGAGGTCTCA-3'.

The allelic makeup of other transacting factors which may be assessedinclude oestrogen and androgen receptors to determine risk ofosteoporosis and/or ischaemic heart disease. The allelic makeup of theandrogen receptor may be also used to assess risk and responsiveness totherapeutic intervention in skin diseases. The allelic makeup of theglucocorticoid receptor and the retinoic acid receptor can be determinedto assess risk of osteoporosis. The allelic makeup of themineralocorticoid receptor can be determined to assess risk ofhypertension and the allelic makeup of proto-oncogenes can be determinedto assess cancer risk. Tissue specific regulators can also be assessedto determine osteoporosis/cancer risk.

In order that the nature of the present invention may be more clearlyunderstood, preferred forms thereof will now be described with referenceto the following examples and figures in which:

FIG. 1 shows lumbar BMD differences in twin pairs according to vitamin Dreceptor alleles.

FIG. 2 shows a map of the vitamin D receptor gene from exon 7 to thestart of the 3' non-coding sequence of exon 9 showing the location ofpolymorphic restriction enzyme sites used in this study and thefragments amplified by PCR used to detect the RFLPs. Asterisk denotespolymorphic site while absence of the asterisk indicates an invariantsite.

FIG. 3 shows bone mineral density is different in VDR genotypes: femalesubjects. Data shows the population mean±standard error mean. p valuesare for the pairwise two-sided Students t-tests for the groups.

FIG. 4 shows the genetic effect on bone mass at the lumbar spine is alsoapparent in males. Symbols are as for FIG. 3.

FIG. 5 shows age related regression of lumbar spine bone mineral densityand intersection with the fracture threshold according to genotype.

FIG. 6 shows age related regression of femoral neck bone mineral densityand intersection with the fracture threshold according to genotype.

FIG. 7 shows bone density differences between twin pairs with respect tozygosity and concordance for VDR alleles. Bone density in the lumbarspine and proximal femur is expressed at the within pair percentdifference in bone density in MZ and DZ twin pairs and according towhether the DZ twin pairs are concordant or discordant for the VDR. TheDZ twins concordant for the VDR alleles are not significantly differentfrom the MZ twins at any site, while the discordant DZ twins aresignificantly different (ANOVA) from both of these groups at each site.The difference between the total DZ group and those concordant for theVDR alleles compared with the MZ twins indicates that 75%; 48%, 59% and90% of the genetic effect can be explained by the VDR alleles at thelumbar spine, femoral neck, Ward's triangle and trochanteric region ofthe proximal femur respectively. Genotype for another developmentaltranscriptional activator, retinoic acid receptor-α (21q7), did notpredict ΔBMD at any site.

FIG. 8 shows the difference in bone density between dizygotic twin pairswith respect to degree of discordance for VDR. The difference in bonedensity between twin pairs is plotted in three groups; 0-completeconcordance, 1-one allele different, 2-both alleles different. Panels A,B, C and D show the analyses for the VDR gene in the lumbar spinefemoral neck. Ward's triangle and trochanteric region respectively.Regression analysis of this effect shows significant relationships atthe lumbar spine (p=0.0001), Ward's triangle (p=0.006) and trochantericregion (p=0.034) and borderline at the femoral neck (p=0.055). Using thesib-pair variance approach, significant relationships were observedbetween the squared difference in bone density within each twin pair(Δ²) and concordance for the VDR gene alleles at the lumbar spine,femoral neck and Ward's triangle and borderline at the trochantericregion of the proximal femur.

    ______________________________________                                        Lumbar spine Δ.sup.2 =                                                                  0.015 + 0.038 * Degree of                                                     discordance (r = 0.43, p = 0.001)                             Femoral neck Δ.sup.2 =                                                                  0.015 + 0.016 * Degree of                                                     discordance (r = 0.29, p = 0.034)                             Ward's triangle Δ.sup.2 =                                                               0.017 + 0.026 * Degree of                                                     discordance (r = 0.34, p = 0.01)                              Trochanteric region Δ.sup.2 =                                                           0.015 + 0.015 * Degree of                                                     discordance (r = 0.27, p = 0.05)                              ______________________________________                                    

FIG. 9 shows higher bone mineral density associated with the b allele ofthe VDR gene.

A. Lumbar spine bone mineral densities of dizygotic twin pairsdiscordant for Bsm-1 alleles (n=22) are plotted as twin and co-twinaccording to genotype. Lines connect bone mineral density values for atwin pair. In 21 of 22 pairs, the twin carrying extra presence of thesite (b) alleles has the higher bone mass (open circles). A single twinpair (black circles) has the reverse situation.

B. Bone mineral density at the lumbar spine amongst unrelatedpremenopausal females according to VDR genotype. One of each(premenopausal female) MZ and DZ twin pair was randomly selected forthis analysis and the numbers of individuals are shown for each group.It is clear that the BB genotype has a lower mean BMD at the lumbarspine while the bb group has the higher mean BMD. The magnitude of thiseffect can be appreciated in relation to the standard deviation of bonedensity in an age-matched population of about 0.11 gm/cm² at each site.The mean ±SE is plotted and significance of the difference betweengroups was calculated by ANOVA. The pair-wise comparisons were made byunpaired Student's-tests. the different groups were not significantlydifferent for age, height or weight.

FIG. 10 shows the results of calcitriol therapy in individuals ofdifferent genotype.

STUDY 1

Methods

Two hundred eighty-eight subjects recruited for epidemiological studiesof bone density were included in the study. All subjects were recruitedfrom the Sydney metropolitan area, latitude 33°52'S, a region of highsunlight incidence. Ninety-one subjects of Caucasian British-Australianorigin (United Kingdom and Irish background) with restriction fragmentlength polymorphism (RFLP) data for the three endonucleases had serumosteocalcin data available. None of the subjects was taking medicationknown to cause bone disease or influence osteocalcin levels. Allsubjects were Caucasian and had normal renal function as determined byserum creatinine.

Serum was collected in the morning after overnight fast, and none of thesubjects was treated with calcitriol prior to venipuncture. Serumosteocalcin was determined by an in-house radioimmunoassay based onrabbit anti ovine osteocalcin (11). The normal range of osteocalcinfound with this assay is 3-18 ng/ml when purified ovine osteocalcin isused. Osteocalcin determinations were made prior to, and independentlyof, the RFLP analysis and the results were stored in a coded fashion.

DNA Analysis. The probe used to identify RFLPs was a 2.1-kilobase-pairfragment of the vitamin D receptor cDNA (3,18) covering the entirecoding region but lacking the 3' untranslated portion of the mRNA.Extraction of DNA from blood and Southern blotting were done by standardmethods. Restriction enzymes were obtained from Pharmacia-LKB and NewEngland Biolabs and used according to the suppliers' specifications.

Statistical Methods. The relative association of the RFLP markers wasassessed statistically for deviation from the null hypothesis of freeassociation by using contingency tables and X² tests. TheStatview-plus-graphics statistical package (Abacus Concepts, Berkeley,Calif.) run on a Macintosh SE/30 computer was used for analysis ofvariance (ANOVA). Fisher's protected least-significant-difference (PLSD)test was used to assess the relationship between RFLP and serumosteocalcin. Significance levels quoted are for the initial F tests onthe null hypothesis (no difference between the means) of the overalleffect and for the confidence level of the pairwise comparison of thecontinuous variable means of each categorical (RFLP) class.

Each RFLP marker system was considered separately for its associationwith osteocalcin serum concentrations by ANOVA comparing categoricalclasses (RLFPs) against the continuous variable (osteocalcin). Theosteocalcin values (ng/ml) were not normally distributed, and sononparametric analysis was performed as well as logarithmictransformation as 1n(1+osteocalcin).

Results

Two previously unreported frequent RFLPs (detected by Bsm I and EcoRV)were found by using the vitamin D receptor cDNA probe, in addition to apreviously reported RFLP detected by Apa I (18). The RFLPs were coded asAa (Apa I), Bb (Bsm I) and Ee (EcoRV), where the uppercase lettersignifies absence of the site and lowercase signifies presence of thesite. The Mendelian nature of the RFLPs was verified by family studies(data not shown). The frequencies of these RFLPs in 266 unselectedvolunteers unrelated to this study are shown in Table 1. The genotypesof 182 individuals were assessed with all three RFLPs (Table 2). Theydemonstrated a strong degree of coassociation, indicating linkagedisequilibrium at this locus. The RFLPs were highly associated such thatAA was found with BB and EE at frequencies of 83% and 92%, respectively;correspondingly, aa was found with bb and ee at frequencies of 61% and72%, respectively. The subsequent functional analysis does not depend onhaplotyping; however, only two of a possible eight haplotypes are neededto account for 53.2% of the test population. The apparent homozygotesdefine the most frequent possible haplotypes as a b e and A B E (Table2).

                  TABLE 1                                                         ______________________________________                                        Frequencies of RFLP Alleles                                                   Enzyme      Allele 1    Allele 2 n*                                           ______________________________________                                        Apa I       A, 0.494    a, 0.506 256                                          Bsm I       B, 0.439    b, 0.560 182                                          EcoRV       E, 0.490    e, 0.510 255                                          ______________________________________                                         *No of individuals tested.                                               

                  TABLE 2                                                         ______________________________________                                        Frequencies of RFLP Genotypes                                                 Homozygotes                                                                              n*         Heterozygotes                                                                             n*                                          ______________________________________                                        aa bb ee   26         Aa Bb Ee    72                                          AA BB EE   19         AA Bb EE    13                                          AA bb EE    2         aa Bb ee     8                                          aa BB ee    2         Aa Bb ee     8                                                                Aa bb Ee     7                                                                Aa bb ee     4                                          ______________________________________                                         *No. of individuals per 182 tested with all three RFLPs (heterozygote         classes with <4 individuals have been excluded).                         

                                      TABLE 3                                     __________________________________________________________________________    Osteocalcin Values and Vitamin D Receptor Alleles                             RFLP n  Median                                                                            Mean                                                                              SD  SE Sig. 1                                                                             Sig. 2                                                                              P value                                     __________________________________________________________________________    Bsm 1                                                                         BB   16 16.8                                                                              2.86                                                                              0.45                                                                              0.11                                                                             BBvsbb                                                                             BBvsBb                                                                              0.0001                                      Bb   46 8.9 2.12                                                                              0.58                                                                              0.09                                                                             0.00005                                                                            0.0001                                            bb   25 8.8 1.97                                                                              0.71                                                                              0.14                                                      Total n                                                                            87                                                                       Apa 1                                                                         AA   25 14.0                                                                              2.53                                                                              0.63                                                                              0.13                                                                             AAvsaa                                                                             AAvsAa                                                                              0.0023                                      Aa   45 9.3 2.18                                                                              0.59                                                                              0.09                                                                             0.001                                                                              0.04                                              aa   20 7.5 1.83                                                                              0.78                                                                              0.18                                                      Total n                                                                            90                                                                       EcoRV                                                                         EE   26 14.0                                                                              2.53                                                                              0.63                                                                              0.12                                                                             EEvsee                                                                             EEvsEe                                                                              0.0153                                      Ee   45 8.8 2.11                                                                              0.60                                                                              0.09                                                                             0.015                                                                              0.015                                             ee   18 10.5                                                                              2.02                                                                              0.83                                                                              0.20                                                      Total n                                                                            89                                                                       __________________________________________________________________________     Osteocalcin values among 91 Caucasian subjects of BritishAustralian origi     (United Kingdom and Irish background) were analyzed with respect to the       three informative RFLPs. As the osteocalcin values were not normally          distributed, they were logarithmically transformed prior to statistical       analysis. Median, median of serum osteocalcin values; Mean, mean of           logtransformed values [In (osteocalcin + 1)]; n, number of subjects; SD,      standard deviation; Se, standard error of the mean; Sig., significance        (probability that such a difference could occur by chance) referring to       the difference between the means of the homozygotes (Sig. 1) and to the       difference between the homozygote (absence of RFLP site) and the              heterozygote (Sig. 2). P value is for the F test on the overall effect.  

                  TABLE 4                                                         ______________________________________                                        Distribution of Subjects with respect to                                      Age, Sex, and Menopausal Status with respect to RFLPs                         Bsm 1          Apa 1        EcoRV                                             BB       Bb     bb     AA   Aa   aa   EE   Ee   ee                            ______________________________________                                        No. of                                                                              16     46     25   25   45   20   26   45   18                          sub-                                                                          jects                                                                         Age,  52 ±                                                                              50 ±                                                                              44 ±                                                                            51 ±                                                                            50 ±                                                                            45 ±                                                                            49 ±                                                                            50 ±                                                                            46 ±                     years 14     14     14   15   15   12   14   15   12                          No.   16     38     20   22   36   18   23   38   15                          female                                                                        Post- 10     18      7   13   17    6   10   22    7                          meno-                                                                         pausal                                                                        Pre-   6     20     13    9   19   12   13   16    8                          meno-                                                                         pausal                                                                        No.    0      8      5    3    9    2    3    7    3                          male                                                                          ______________________________________                                    

The relationship between RFLPs and serum osteocalcin was analyzed in the91 normal subjects with serum osteocalcin data (Table 3). Thedistribution of this population with respect to age, sex, and menopausalstatus is shown in Table 4. Age was not significantly related to anyRFLP genotype. The osteocalcin levels of the Bsm I BB group aresignificantly higher than those of the Bsm I bb group (P=0.0001). Theother RFLPs show the same effect with highly significant P values forthe Apa I allele system (AA versus aa, P<0.0025) and a weaker P valuefor the EcoRV RFLP (EE versus ee, P=0.015). With all three RFLPs theabsence of restriction-site alleles (A, B, E) is associated with highosteocalcin levels and the presence of restriction-site alleles (b, a,and e, respectively) with low osteocalcin levels: BB, 16.8 ng/ml; Bb,8.9 ng/ml; and bb, 8.8 ng/ml (medians). Nonparametric statisticalanalysis (Kruskal-Wallis) of raw osteocalcin values gave essentially thesame results as ANOVA: Apa I, P-0.0016; Bsm I, P=0.0001; EcoRV,P=0.0044.

Since the Bsm I and Apa I RFLPs were the most predictive, the populationwas subdivided according to the nine possible combinations of thesealleles. This produced a clear separation of the serum osteocalcinvalues according to genotype (FIG. 1). Since the weaker association ofthe EcoRV marker may be determined by its disequilibrium with the othermarkers, we examined the distribution of Apa I and Bsm I alleles andosteocalcin values within individuals with the EE genotype (FIG. 2). TheBsm I marker essentially dictated the inferred haplotypes and theirassociated osteocalcin values (P=0.003).

The genotype prediction of serum osteocalcin levels was maintained forBsm I and Apa I when males (n=14) were excluded (Bsm I, P=0.0001; Apa I,P=0.0034; ANOVA values for the overall effect). Menopause has beenassociated with an increase in osteocalcin values, with a wide variationin osteocalcin values being observed in the early postmenopausal years(19-21). Therefore the role of menopausal status was assessed bymultiple regression analysis and analysis of covariance including age,menopausal status, and Bsm I genotype. Menopausal status was a weakerdeterminant of serum osteocalcin concentrations than Bsm I polymorphism(r=-0.44, P<0.001). Two-factor ANOVA yielded the same result; Bsm I,P=0.0002; menopausal status, P=0.24. Analyzing premenopausal andpostmenopausal women separately did not alter the results, and genotypewas a stronger predictor than menopausal status (FIG. 1).

STUDY 2

Materials and Methods

Subjects

Subjects were 535 unrelated volunteers (447 females and 88 males) whohad enrolled in studies of the effect of genetics on bone density. Thesubjects were obtained from requests through the media in the Sydneymetropolitan area. The mean ages of the subjects were 51.4±13.8 yr(mean±SD; range 20-84 yr) for females and 40.6±16.0 yr (20-79 yr) formales. Subjects in this analysis were of Caucasian British-Australianorigin (United Kingdom and Irish background). Menopausal status wasconfirmed by the presence of elevated FSH and LH and low estradiollevels, with an absence of menses for at least 12 months. Subjects witha history of bone disease, illness, bilateral ovarectomy or drug use(including hormone replacement therapy) which could affect bone turnoverand bone density were excluded from this study.

Bone Mineral Density Analysis

Bone mineral density (BMD), expressed as an area density in g/cm², wasmeasured in the lumbar spine (L2-4) and femoral neck using either dualphoton absorptiometry or dual energy x-ray absorptiometry (Lunar DP3 orDEXA, respectively, Lunar Radiation NCo. Madison, Wis.) as previouslydescribed (Pocock et al. 1987).

DNA Analvsis; PCR (Polymerase chain Reaction) and RFLP Analysis usingEndonuclease Digestion

Blood was collected into heparin treated tubes and leukocytes separatedby sedimentation trough physiological saline solution in a clinicalcentrifuge. Purified leukocytes were lysed in leukocyte lysis buffer (10mM Tris-HCl, pH7.4, physiological saline and 0.5% w/v sodium dodecylsulphate). Lysate was treated with proteinase K (Applied Biosciences,Palo Alto U.S.A.) at 50 ug/ml for 2 hour at 65 Celsius. DNA wasextracted by repetitive phenol chloroform solvent extraction asdescribed in Maniatis et al. and ethanol precipitated prior. DNA wasredissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) andquantitated by ultraviolet absorbance at 260 Nm.

The vitamin D receptor gene from exon 7 to the 3'-untranslated regionwas sequenced. The sequence is set out in Table 5, (SEQ ID:NO1).

Four oligonucleotide primers were synthesized to amplify the 3' flankingregion of the VDR gene. Detection of the Bsm1 site was facilitated byamplifying a region spanning the site, with one primer originating inexon 7(5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3') and the other in intron8(5'-AACCAGCGGAAGAGGTCAAGGG-3') producing a 825 base pair fragment.Detection of ApaI and TaqI sites was facilitated using a singleamplification one

                                      TABLE 5                                     __________________________________________________________________________    (SEQ ID NO: 1)                                                                __________________________________________________________________________    Sequence Range: 1 to 2169                                                      ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                                    __________________________________________________________________________     Primer underlined on top strand is a foward primer, those on the bottom       strand are reverse primers.                                                   Any pair wise combination of these primers or primers based on this and       surrounding sequence can amplify the region by polymerase chain reaction.                                                                              

primer in intron 8(5'-CAGAGCATGGACAGGGAGCAAG-3') and the other in exon 9(5'-GCAACTCCTCATGGCTGAGGTCTCA-3' producing a 740 base pair fragment(FIG. 2).

PCR was carried out in a volume of 20 ul containing 200ng genomic DNA,20 pmol of each primer, 200 uM dNTPs, 50 mM KCl, 10mM Tris (pH8.3), 1.5mM, MgCl₂ and 1 U Taq DNA polymerase (TOYOBO, Osaka, Japan). Each samplewas subjected to 37 amplification cycles as follows: Step 1 -3 min at94° C., 1 min at 62° C., 2 min at 72° C.; Step 2 to 6 -20 sec at 94° C.,20 sec at 62° C., 1 min at 72° C., Step 7 to 36 -5 sec, 5 sec, 30 secrespectively. Amplification regimes should be optimised for anyparticular thermal cycling device. A 10 ul aliquot of each PCR productwas digested with 5 units of endonuclease Bsm1 at 65° C. (New EnglandBiolabs, Massachusettes U.S.A.), Apa1 at 37° C. or Taq1* (Promega Co.Australia) at 65° C. for 1 hour. A clone of an unrelated gene was usedas an internal control for both Bsm1 and Apa1 digestion. For Taq1digestion, an invariant Taq1 site in the PCR product itself was used asan internal control. The digested PCR products were separated on 1.2%(Bsm1 and Apa1), or 2.0% (Taq1) agarose gels containing 0.5 ug/mlethidium bromide, 0.09M Tris-Borate and 0.002M EDTA, pH 8.3 for 1 hr at100 V. EcoRI digested SPP1 marker (Bresatec Limited, Adelaide,Australia) was used as the size standard for all agarose gels. Due tothe sequence of the relevant sites several other restriction enzymes canbe used to detect these polymorphisms. Bsm1 site sequence from aninvariant adjacent Stu-1 site; B allele AGGCCTGC GCATTCCC (SEQ ID:NO 6),b allele underlined G is an A. This sequence change can be detected withAos1, Fsp1, Mst1, Fdi2, Hinp1, Hha1 and their isoschizomers. Sequence atthe polymorphic Apa1 site ending in an adjacent invariant Pvu2 site is:A allele GAGG GGCCCAGCTG (SEQ ID:NO 7), in the a allele the underlined Gis a T. The presence of the G can be detected by Ban2, Aoc2, Pss1, Pa11,Hae3, Cfr3I, Asul, Sau96I, Eco0109I, Dra2, and isoschizomers. Thepresence of the T creates a polymorphisms for Ban1, and itsisoschizomers. The sequence of the Taq1 polymorphism spanning invariantHba1 to Hae3 sites is: T allele GCGCTGAT TGAGGCC (SEQ ID:NO 8), in the tallele the underlined T is a C. This polymorphism can be also detectedby Mbo1, Sau3A, Dpn1 and their isoschizomers.

Taq1* RFLP: We have previously reported that Bsm1 and Apa1 RFLPs in thevitamin D receptor gene predict serum osteocalcin levels. Thesepolymorphic sites are located in the region of genomic DNA from exon 7to the 3' untranslated region (3'-UTR). To characterize the differencesbetween two common vitamin D receptor gene alleles (AB and ab), we havesequenced this region in homozygotes of genotypes AABB, aabb. We haveidentified a number of sequence differences, including 15 non codingchanges. There is a single synonymous coding region change, a T for C inan isoleucine codon (ATT to ATC, isoleucine codons) in exon 9.

Statistical Analysis

Analysis of the variance (ANOVA) was performed using Statview+Graphicsstatistical package (Abacus Concepts, Berkeley, Calif., U.S.A.) on aMacintosh SE/30 computer. Fisher's protectedleast-significant-difference (PLSD) test was used to assess therelationship between RFLP and the BMD, height, weight. Significancelevels quoted are for the initial F tests on the null hypothesis (nodifference between the means) of the overall effect and for theconfidence level of the pairwise comparison of the continuous variablemeans of each categorical (RFLP) class. Students t-test was used forpairwise comparisons. Relationships of continuous and categoricalvariables were established by multiple regression. Relationships betweenRFLP markers were established by contingency tables and Chi square.

Results

The frequencies of these three RFLPs in 535 subjects are shown Table 6.The RFLPs were coded as Bb (Bsm1), Aa (Apa1) and Tt (Taq1), where theuppercase letter signifies absence of the site and lowercase signifiespresence of the site. The frequencies of Bsm1 and Apa1 RFLP are similarto that set out above (Table 1). RFLPs had a high degree ofcoassociation (Table 7). The AA genotype is highly associated with BBand tt at frequencies 92.7% and 95.3%, respectively; correspondingly, aawas found with bb and TT at frequencies of 61.6% and 65.3%,respectively. Comparing Bsm1 with Tag1 RFLP, tt, Tt, and TT genotypesare highly associated with BB, Bb and bb at frequencies 95.5%, 95.1% and96.4% respectively. Because the Bsm1 and Tag1 results are so closelycorrelated, in subsequent discussions we have equated Bsm1 and Tag1results and will refer only to Bsm1 results.

The relationship between RFLPs and BMD at both LS and FN sites wereanalyzed in the 535 subjects. The distribution of this population withrespect to age, height, weight, and menopausal status is shown in Table8. Age, height, and weight were not significantly related to any RFLPgenotype (Table 9). In females, mean LS BMD of the BB and AA group are9.9% (1.017 vs 1.118) and 8.6% (1.049 vs 1.139) lower than those of thebb and aa groups respectively. The FN BMD of the BB and AA groups arealso 5.6% and 5.3% lower than those of the bb and aa groupsrespectively. A heterozygote effect indicating co-dominance of alleleswas also observed (FIG. 3). Lower LS and FN BMD were associated with theabsence of both restriction site alleles (BA). The differences of meanBMD at LS and FN between BBAA genotype and bbaa genotype was wider

                  TABLE 6                                                         ______________________________________                                        Frequencies of RFLPs in study population.                                     Genotype   N      Frequency %    Allele                                       ______________________________________                                        BB          89    16.8           B = 0.418                                    Bb         266    50.1           b = 0.582                                    bb         176    33.1                                                        AA         133    25.5           A = 0.512                                    Aa         268    51.3           a = 0.488                                    aa         121    23.2                                                        TT         188    35.4           T = 0.596                                    Tt         257    48.4           t = 0.404                                    tt          86    16.2                                                        ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        RFLP markers have a high degree of coassociation.                             Bsm-1 genotypes tabulated with Apa-1 and Taq1                                 genotypes. n refers to number of individuals. Chi                             square value and p value reflect the rejection of the                         null hypothesis of no association between the markers.                        Marker      BB     Bb         bb   total n                                    ______________________________________                                        AA          78      44         11  133                                        Aa          5      206         56  267                                        aa          4       8         106  118                                        total n     87     258        173  518                                         Chi.sup.2 = 428                                                               p = 0.0001                                                               

    TT           1      14        173  188                                        Tt           5     249         3   257                                        a           83      3          0    86                                        total n     89     266        176  531                                        ______________________________________                                         Chi.sup.2 = 912                                                               p = 0.0001                                                               

                  TABLE 8                                                         ______________________________________                                        Population characteristics of study group                                     Sex              Number                                                       ______________________________________                                        Males             88                                                          Females          447                                                          Premenopausal    185                                                          Postmenopausal   262                                                          Mean years since 11.3 ± 0.07                                               menopause ± SEM                                                            ______________________________________                                        Mean values of anthropomorphic parameters in                                  total subjects (±SEM).                                                     ______________________________________                                        Age (year)       49.6 + 0.6                                                   Height (cm)      163.8 + 0.4                                                  Weight (kg)      64.8 + 0.5                                                   ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Mean values of anthropomorphic parameters                                     according to Bsm-1 genotype                                                   Genotype   n      Age        Height Weight                                    ______________________________________                                        Females                                                                       BB          75    50 + 2     162 + 1                                                                              62 + 1                                    Bb         216    51 + 1     161 + 1                                                                              63 + 1                                    bb         154    52 + 1     161 + 1                                                                              64 + 1                                    p value           0.6        0.3    0.9                                       Males                                                                         BB         14     35 + 4     177 + 2                                                                              72 + 10                                   Bb         50     41 + 2     176 + 1                                                                              75 + 10                                   bb         22     42 + 4     176 + 2                                                                              74 + 14                                   p value           0.9        0.6    0.4                                       ______________________________________                                    

(13.4%, 7.8% respectively) than those of BB and bb or AA and aa (Table9).

The effect of genotype was assessed by multiple regression analysis ofcovariance including age(yr), menopausal status (year post menopause;YPM), height (cm), weight (kg) and Bsm1 genotype (BB=1, Bb=2, bb=3) infemales, giving the equation; LS BMD (g/cm2)=0.419+0.054 Bsm1 genotype-0.004 age -0.994 YPM+0.02 weight+0.004 height (n=425) r=0.58 R2=0.34,

    ______________________________________                                        Bsm1         age      YPM      Weight Height                                  ______________________________________                                        p value 0.0001   0.0013   0.0001 0.0045 0.003                                 F-score 24.9     16.0     10.4   8.2    8.9                                   ______________________________________                                    

FN BMD g/cm2)=0.456+0.025 Bsm1 genotype -0.004 age -0.004 YPM+0.04weight+0.02 height (n=425) 4=0.68, R2=0.47

    ______________________________________                                        Bsm1         age      YPM      Weight Height                                  ______________________________________                                        p value 0.002    0.0001   0.0004 0.0001 0.022                                 F-score 9.6      41.7     12.8   35.2   5.3                                   ______________________________________                                    

Both lumbar spine and femoral neck BMD were negatively and independentlycorrelated with-the menopausal status, age, Bsm1 RFP was also correlatedindependently with BMD at LS and F in females. Male's results were asfollows:

LS BMD (g/cm2)=1.039+0.058 Bsm1 genotype (n=85)

r=0.22 RS=0.05, p=0.038, F-score 4.9

FN BMD(g/cm2)=1.046-0.003 age (n=85)

r=0./32, R2=0.10, p=0.017, F-score 5.9

Intercept with the Fracture Threshold

A value of lumbar spine BMD, below which a heightened risk ofosteoporotic fracture exists, was derived form a large cross sectionalstudy in the city of Dubbo Australia. This value 0.97 gm.cm² is similarto a fracture threshold described from an American population. Clearly,if VDR genotype affects BMD and subsequently osteoporosissusceptibility, a difference in the intercept of the age related changein bone mass and the fracture threshold should be apparent betweengenotypes. FIG. 5 shows simple age related regression lines for femaleLS BMD of BB, Bb and bb genotypes intersecting the fracture thresholdvalue. A comparison between BB and bb reveals a 10 year difference inthe intercept (60.3 yr versus 71.1 year, respectively) with anintermediate value for the Bb heterozygotes (68.1 year). A similarresult was apparent for the neck of femur (FIG. 6) using a fracturethreshold of 0.7 gm/cm² (BB, 66 years; Bb, 70 years; bb, 74 years).

STUDY 3

The effect of the common VDR gene alleles on bone density was examinedusing the twin model, in which within-pair comparisons eliminate age andvarious cohort confounders. 250 Caucasian twins were studied comprising70 MZ and 55 DZ twin pairs, including 7 male MZ pairs and 6 male DZpairs, aged between 17 and 70 years; MZ 45±13 yrs and DZ 44±11 yrs,mean±SD. Bone density was measured at the lumbar spine and proximalfemur with a Lunar DP3 dual-photon absorptiometer (LUNAR Corporation,Madison, Wis.) or Lunar DEXA dual energy X-ray absorptiometry aspreviously described (Pocock et al 1987). All female twin pairs wereconcordant for menopausal status and if post menopausal, for years sincemenopause.

The VDR gene in the region bearing the polymorphic sites for the Bsm-1,Apa-1 and EcoRV sites previously shown to predict differences in boneturnover markers was sequenced. These sites are in the region of thegene from exon 7 to the 3'-UTR. None of the polymorphic sites was in thecoding region or involved potential splice sites and the highlyinformative Bsm-1 site was found to arise from a G for A substitution inintron 8. There was only one difference in the coding region between thetwo most common allelic forms. This included a T for C substitution inexon 9, changing ATT to ATC, without changing the encoded amino acidsequence (isoleucine). The DNA sequence flanking the Bsm-1 site was usedin a polymerase chain reaction-based method to amplify a 2.1-2.2 kbfragment from exon 7 to exon 9 to facilitate genotyping of subjects. PCRamplification of leucocyte DNA was performed with a Corbett FTS-1Thermal Sequencer (Corbett Research, Mortlake NSW, Australia) PCRinstrument using primers

5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' and

5'-AACCAGCGGGAAGAGGTCAAGGG-3' prior to endonuclease digestion with Bsm-1(New England Biolabs Inc, Gene Search, Brisbane, Australia). Thepresence of the Bsm-1 site cuts a 825 bp product to 650 bp and 175 bpfragments. A 4.7 kb plasmid with a single Bsm-1 site, which lineariseswith Bsm-1 digestion, was used as an internal control to avoidmisassignment of the allelic forms due to partial digests.

From twin studies the within pair difference in BMD (ΔBMD %) at thelumbar spine and proximal femur was examined in relation to allelicvariation in DZ twin pairs (FIG. 7). In both regions this wassignificantly less in DZ twins concordant compared to those discordantfor VDR gene alleles. The ΔBMD % for lumbar spine in the MZ twins wasnot significantly different from that in the DZ twins concordant for theVDR alleles, both of which were statistically different from those in DZtwins discordant for the alleles (p<0.0001). Similar but weaker effectsin the proximal femur are consistent with stronger environmentalinfluences on bone density in this region. Limiting the analysis topremenopausal twins did not alter the results. Controlling for potentialconfounding by anthropomorphic features of height and weight, VDRgenotype remained the strongest predictor at the lumbar spine (p=0.0002)and the trochanteric region (p=0.02) although not at the neck region ofthe proximal femur. In view of the previously demonstrated co-dominanteffect of Bsm-1 alleles on bone turnover indices, we would expect aco-dominant effect on the bone mass trait with a linear relationshipbetween the degree of difference in genotype and the difference in traitwithin twin pairs (FIG. 8). According to the sib-pair linkage analysisapproach, a significant correlation between the squared difference in atrait and the proportion of identical genes within a sibling pairindicates genetic linkage. By this analysis the VDR gene alleles wereco-dominant at the lumbar spine and most sites in the proximal femur.Comparing the Δ BMD % with respect to degree of concordance for VDRalleles showed 1.5 to 2.5-fold greater within pair differences for thediscordant twins (see FIGS. 7 and 8). In 21 of 22 dizygotic twin pairsdiscordant for the VDR alleles, the b allele was associated with higherbone density (FIG. 9A). In premenopausal females (randomly selected assingletons from MZ and DZ twin pairs), the VDR bb genotype was alsoassociated with higher bone density (FIG. 9B) while the BB genotype wasassociated with lower bone mass with a clear codominant effect betweenthe alleles (FIG. 9B).

These data demonstrate that the differences of VDR gene alleles indicatea major proportion of the differences in bone density in a population ofnormal individuals. The BB,AA,EE and/or tt VDR genotypes are associatedwith low BMD in both females and males. VDR gene RFLPs genotypes aretherefore useful predictors of propensity to high bone turnover and lowbone mass, physiological variability not only in peak bone mass but alsobone mass in later life in both females and males. Until now, themechanisms of the genetic effects on bone density and bone turnover havebeen unclear. However, 1,25-dihydroxyvitamin D is a enhancer ofosteocalcin synthesis through the vitamin D responsive element in thepromotor of the VDR gene (Morrison 1989 Science). The present inventorshave also shown that common allelic variants of the VDR gene areassociated with differences in the serum osteocalcin levels. Moreover,these allelic variants of the VDR gene predict the difference in bonedensity between dizygotic twin pairs.

It is concluded that these VDR gene RFLP's are markers for physiologicalvariability in bone mass in both females and males. The presentinventors have found that Bsm1 RFLP correlated independently with BMD atLS and FN.

                  TABLE 10                                                        ______________________________________                                        Age and years since menopause (YSM)                                           amongst twins. A, those DZ twins concordant and                               discordant for VDR gene alleles; B; individuals with                          differing alleles for the VDR gene. All values are                            expressed as means ± SD.                                                   ______________________________________                                                     Concordant                                                                              Discordant                                             ______________________________________                                        Age          41.1 ± 10.6                                                                          45.5 ± 12.3                                                      n = 30    n = 23                                                 YSM          4.4 ± 3.6                                                                            9.3 ± 5.3                                                        n = 5     n = 7                                                  ______________________________________                                               BB            Bb        bb                                             ______________________________________                                        Age    44.0 ± 12.8                                                                              43.6 ± 13.4                                                                          45.7 ± 11.3                                 YSM    9.0 ± 2.5  11.8 ± 9.5                                                                           9.8 ± 7.8                                          n = 9         n = 12    n = 7                                          ______________________________________                                    

                  TABLE 11A                                                       ______________________________________                                        Correlation co-efficients between monozygotic                                 and dizygotic twin pairs with dizygotic pairs                                 further segregated into those concordant and                                  discordant for vitamin D receptor genotype.                                                                rDZ con-                                                                             rDZ dis-                                  Variable                                                                              rMZ    rDZ    p      cordant                                                                              cordant                                                                              p                                  ______________________________________                                        Lumbar  0.81   0.16   <0.0001                                                                              0.41   0.04   <0.001                             Spine                                                                         Femoral 0.79   0.43   <0.0008                                                                              0.44   0.41   NS                                 Neck                                                                          Ward's  0.83   0.51   <0.004 0.43   0.54   NS                                 Triangle                                                                      Tro-    0.82   0.34   <0.0002                                                                              0.49   0.38   <0.006                             chanteric                                                                     Weight  0.80   0.42   <0.0004                                                                              0.36   0.45   NS                                 ______________________________________                                         Notes:                                                                        p; denotes the p value for the test that correlation coefficients are         significantly different. In the twin model a significant difference           between rMZ and rDZ is evidence for a genetic effect on the trait in          question. In our comparison of DZ twin pairs with the same withinpair VDR     genotype (DZconcordant) or different withinpair VDR genotype                  (DZdiscordant), a significantly different correlation between                 rDZconcordant and rDZdiscordant is supportive of a contribution of the VD     genotype to the genetic effect on the trait in question.                 

                  TABLE 11B                                                       ______________________________________                                        Within-twin pair proportional difference in bone mineral                      density according to zygosity and degree of discordance of the                twin pair of alleles of the vitamin D receptor gene. Values                   of percentage differences are means ± sem.                                                                        Tro-                                                Lumbar    Femoral Ward's  chanteric                              Variable                                                                             n     spine     Neck    Triangle                                                                              region                                 ______________________________________                                        MZ     69     6.0 ± 0.7                                                                            8.1 ± 0.9                                                                         10.0 ± 1.2                                                                          9.3 ± 1.0                          DZ-all 55    11.9 ± 1.4                                                                           12.1 ± 1.4                                                                         15.9 ± 2.0                                                                         13.5 ± 1.7                          DZ-con-                                                                              33     7.5 ± 1.3                                                                           10.2 ± 1.5                                                                         12.4 ± 2.0                                                                          9.7 ± 1.7                          cordant                                                                       DZ-dis-                                                                              22    18.5 ± 2.5                                                                           15.1 ± 2.8                                                                         20.6 ± 3.8                                                                         18.7 ± 3.0                          cordant                                                                       Genetic      75%       48%     59%     90%                                    effect                                                                        DZ-dis-                                                                       cordant:                                                                      -1 allele                                                                            14    15.6 ± 2.5                                                                           13.2 ± 2.7                                                                         16.4 ± 3.7                                                                         19.9 ± 2.8                          -2      8    22.0 ± 5.2                                                                           17.9 ± 5.9                                                                         28.5 ± 8.2                                                                         16.4 ± 7.0                          alleles                                                                       DZ-dis-      2.47      1.48    1.66    1.93                                   cordant/                                                                      DZ-con                                                                        DZ-2         2.93      1.75    2.30    1.71                                   alleles/                                                                      DZ-con                                                                        ______________________________________                                    

Importantly, the homozygous BBAA or AAtt genotype are associated withlow bone density, and mean BMD at the LS and FN site were about 12% and8% lower in BBAA homozygotes compared with bbaa genotype in both femalesand males. These genotypic differences are important for later life,because these differences of BMD indicate a 10 year difference in thefracture threshold. These allelic differences provide a mechanism forthe genetic effect on bone mass observed in twin studies and provide asimple genetic test of carrier status for low bone mass alleles.Identification of the vitamin D receptor genotype as an importantdeterminant of bone mass may open new avenues for prevention and therapyfor osteoporosis.

Demonstration of Differences in Response to Treatment in Different GeneTypes.

Data described above have demonstrated that the VDR alleles describedabove are functionally different. It would therefore be expected thatindividuals of different genotype would exhibit different responses totreatment with calcitriol and/or analogues. This was confirmed byexamining responses to calcitriol administration in 10 normal youngfemales of each homozygous Bsm 1 genotype (BB and bb) and analysingresponses to treatment in three markers of bone calcium metabolism;osteocalcin, parathyroid hormone and urinary calcium (see FIG. 10).

Osteocalcin serum levels were different (p<0.01) at basline in the BBand bb groups. The BB genotype again had the higher osteocalcin. Aftercalcitriol treatment the bb group had a higher percent response frombaseline than the BB group. Although the BB group had a lesser percentresponse, since they had a higher baseline osteocalcin, the totalresponse was higher.

Parathyroid hormone is known to be repressed by calcitriol, however, theextent of repression by calcitriol treatment was significantly differentin the two genotypic groups. Parathyroid hormone was weakly repressed inthe BB group and strongly repressed in the bb group, indicatingsubstantial differences in the response of PTH to calcitriol therapy.Total urinary calcium excretion over the treatment period (area underthe curve) was significantly higher in the BB group than the bb groupindicating different calcium handling responses according to genotype.the reduced repression of parathyroid hormone in the face of calcitrioltreatment, coupled with increased urinary calcium output indicatesdifferent calcium homeostatic mechanisms, compatible with mobilisationof skeletal calcium. VDR and Other Conditions

The vitamin D receptor and vitamin D endocrine system are implicatedinseveral other pathological and physiological states. Such differences inthe vitamin D receptor gene, leading to different responses toendogenous calcitriol, exogenous calcitriol and therapy using vitamin Danalogues, will also result in differences in progression andsusceptibility to other disorders where a significant component ofregulation is effected by calcitriol. Known examples of conditions anddiseases where the vitamin D endocrine system and VDR mediated eventsoccur include AIDS virus (HIV-1) replication, breast cancer cellproliferation, colonic cancer cell growth, keratinocyte differentiation,psoriasis cell replication and function, spermatogenesis, melanoma andother tumours.

As a result of the invention described herein, it is therefore obviousthat functionally different alleles of the VDR could affect thesusceptibility, progress, prognosis and therapeutic efficacy of varioustreatments, in such diseases and conditions where the vitamin D receptorand vitamin D endocrine system are known to regulate aspects of thedisease process. While these are examples of physiological and diseaseprocesses influenced by the vitamin D endocrine system, it is in no wayexclusive of other processes influenced by the vitamin D endocrinesystem. Given the data described herein, it is obvious that allphysiological and disease processes known to be influenced by thevitamin D endocrine system, as described in a recent comprehensivereview by Walters, M. (newly identified actions of the vitamin Dendocrine system; Endocrine reviews, 13:719-764) and papers referred totherein, could be assessed and investigated in the way described herein,and that these could be influenced by the vitamin D receptor genotypeand therefore the genotype of an individual will be of importance to theprognosis, progression, susceptibility and treatment of all conditionsand diseases in which vitamin D receptor and the vitamin D endocrinesystem are involved.

Irrespective of the physiological mechanism, these data have identifiedfor the first time a gene involved in the regulation of bone density.Importantly the magnitude of the effect is such that it explains themajority of the strong genetic effect on bone density and indeed morethan half of the adjusted population variation in bone density. Thesefindings, which will allow earlier interventions in those at increasedrisk of osteoporosis, provide important insight into the mechanism ofthe wide population variance in bone density and open the way todevelopment of novel specifically targeted therapies. This single genewith pleiotropic transcriptional activities is a model for manypathophysiological processes previously considered subject to complexmulti-factorial genetic regulation.

This study describes a functional definition of naturally occurringalleles of a trans-acting transcriptional activator by correlation withthe product of a target gene. The data also indicate that the receptorallelic differences also relate to major differences in a targetorgan--i.e., bone density. This method of genetic analysis provides aparadigm for the investigation of the functional significance of naturalallelic variation within the genes of the ligand-activated receptorsuperfamily, which can contribute substantially to a more completeunderstanding of the steroid hormone endocrine system. It is alsoapplicable to the genes for trans-acting regulators of all kinds.

Genotypic variations in transcriptional regulators of genes encodingregulatory and/or structural proteins, determine physiologicalset-points and predisposition to pathophysiological states withimplications for susceptibility to disease and for determining likelyresponses to therapy. These genotypic variants are a general model foruse in the determination of disease risk and for choice of therapy inprevention and treatment.

As a specific example of this model, allelic variants in the vitamin Dreceptor gene determine bone turnover, bone mass and sensitivity toenvironmental factors. As such these variants are markers of risk ofdevelopment of osteoporosis and indicate likely response to variousmodalities of therapy.

The inventors have identified RFLP markers that define functionallydifferent vitamin D receptor alleles. The RFLPs herein described arephysical markers that are linked to genetic phenomena. The inventorsadvise that it is now obvious that any other RFLP, physical marker,polymorphic sequence, or genetic effect detectable in the vitamin Dreceptor gene or flanking DNA, which is in linkage with the currentlydefined markers, could provide the same information content as themarkers herein described, dependent on the extent of linkage between themarkers defined herein and any other such marker, consisting of RFLP,physical, polymorphic sequence, or genetic effect. The inventors therebystate that other markers, known or unknown, in linkage with the markersherein described, represent a claimed usage of this invention.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

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7. Hughes, M. R., Malloy, P. J., Kjeback, D. G., Kesterson, R. A., Pike,J. W., Feldman, D & O'Malley, B. W. (1988) Science 242, 1702-1705.

8. Lehrer, S., Sanchez, M., Song, H. K., Dalton, J., Levine, E.,Savoretti, P., Thung, S. N. & Schachter, B. (1990) Lancet 355, 622-624.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2169 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CAACCAAGACTACAAGTACCGCGTCAGTGACGTGACCAAAGGTATGCCTAGACTCCACCT60                CCTGGGGAGTCTTTTTCAGCTCCCAGATTCTGGCTCCACCCGTCCTGGGGTTTGGCTCCA120               ATCAGATACATGGGAGGGAGTTAGGCACCAACAGGGAGAGAAGGGCGAGGGTCAGACCCA180               TGGGGTTGGAGGTGGGTGGGCGGCTCCTCAGCTCTTGCCCGCAGTACCTGGCCATTGTCT240               CTCACAGGCCGGACACAGCCTGGAGCTGATTGAGCCCCTCATCAAGTTCCAGGTGGGACT300               GAAGAAGCTGAACTTGCATGAGGAGGAGCATGTCCTGCTCATGGCCATCTGCATCGTCTC360               CCCAGGTATGGGGCCAGGCAGGGAGGAGCTCAGGGACCTGGGGAGCGGGGAGTATGAAGG420               ACAAAGACCTGCTGAGGGCCAGCTGGGCAACCTGAAGGGAGACGTAGCAAAAGGAGACAC480               AGATAAGGAAATACCTACTTTGCTGGTTTGCAGAGCCCCTGTGGTGTGTGGACGCTGAGG540               TGCCCCTCACTGCCCTTAGCTCTGCCTTGCAGAGTGTGCAGGCGATTCGGTAGGGGGGAT600               TCTGAGGAACTAGATAAGCAGGGTTCCTGGGGCCACAGACAGGCCTGCGCATTCCCAATA660               CTCAGGCTCTGCTCTTGCGTGAACTGGGCTCAACATTCCTGTTATTTGAGGTTTCTTGCG720               GGCAGGGTACAAAACTTTGGAGCCTGAGAGATGGTTCTGCCTATATAGTTTACCTGATTG780               ATTTTGGAGGCAATGTGCAGTGACCCTTGACCTCTTCCGCTGGTTAGAGGTGAGAAGAGG840               GAGAAAAGGCCGAAGAGAAGTTATTGTGACCTTGGGACATGATGTCGGTGATGAGGTCCA900               AAGAGGGGCGGCCCTGCCTCAGCCTGTGCTAGTGGCCTGTGCCCAGGGATGCTTTCCTGG960               ACTGGAGGCTCAAGGAATGGAGATGGCTCCTCTACCCCTGCCCAGCCAGCCTTCTCTCAT1020              TCATTCATCCACTTAGCAACAATTTATTGAGCACCTATTAGGTACCAGGCACTATGCTAG1080              GTACTGGGGTTCAGCAGCAAATGGGACACAGGCTCCTCTCCCATGAAGCTTAGGAGGAAA1140              CATTAAACAAATGTTATTTAATTATTAATTCCTAACAAGGCAAGAGTTTTAAAAATAAAG1200              TAAGTGATGCTACAGAAGGGTAGAATAGAAGGAGGGAAGCTGACGTGGTCTGGGCTACAG1260              AGGTAGAGTGTTGCCAGGAATGGCCTTTTGGAGGAAGACCTTTTGAGCTGTTATCCAAAG1320              GATCAGTAAGAGTCTGGCAAAGATAGCAGAGCAGAGTTCCAAGCAGAGGGAGCACAGATG1380              TGAAGGCTGGTGGCAGAGAGCATGGCGCATCGGGTCGCTGAGGGATGGACAGAGCATGGA1440              CAGGGAGCAAGGCCAGGCAGGGACAGGGCCAGGTGCGCCCATGGAAGGACCTAGGTCTGG1500              ATCCTAAATGCACGGAGAAGTCACTGGAGGGCTTTGGGGCCAGGCAGTGGTATCACCGGT1560              CAGCAGTCATAGAGGGGTGGCCTAGGGGGTGCTGCCGTTGAGTGTCTGTGTGGGTGGGGG1620              GTGGTGGGATTGAGCAGTGAGGGGCCCAGCTGAGAGCTCCTGTGCCTTCTCTACTCCCGT1680              GCCCACAGATCGTCCTGGGGTGCAGGACGCCGCGCTGATTGAGGCCATCCAGGACCGCCT1740              GTCCAACACACTGCAGACGTACATCCGCTGCCGCCACCCGCCCCCGGGCAGCCACCTGCT1800              CTATGCCAAGATGATCCAGAAGCTAGCCGACCTGCGCAGCCTCAATGAGGAGCACTCCAA1860              GCAGTACCGCTGCCTCTCCTTCCAGCCTGAGTGCAGCATGAAGCTAACGCCCCTTGTGCT1920              CGAAGTGTTTGGCAATGAGATCTCCTGACTAGGACAGCCTGTGCGGTGCCTGGGTGGGGC1980              TGCTCCTCCAGGGCCACGTGCCAGGCCCGGGGCTGGCGGCTACTCAGCAGCCCTCCTCAC2040              CCGTCTGGGGTTCAGCCCCTCCTCTGCCACCTCCCCTATCCACCCAGCCCATTCTCTCTC2100              CTGTCCAACCTAACCCCTTTCCTGCGGGCTTTTCCCCGGTCCCTTGAGACCTCAGCCATG2160              AGGAGTTGC2169                                                                 (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAACCAAGACTACAAGTACCGCGTCAGTGA30                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AACCAGCGGAAGAGGTCAAGGG22                                                      (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CAGAGCATGGACAGGGAGCAAG22                                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCAACTCCTCATGGCTGAGGTCTCA25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGGCCTGCRCATTCCC16                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAGGKGCCCAGCTG14                                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "OLIGONUCLEOTIDE"                                    (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GCGCTGATYGAGGCC15                                                             __________________________________________________________________________

We claim:
 1. A method of assessing an individual's predisposition to lowor high bone density, development of high or low bone turnover and/orresponsiveness to therapy for conditions relating to bone density orbone turnover comprising analyzing allelic variation in the vitamin Dreceptor gene of the individual, thereby determining an individual'spredisposition to low or hiqh bone density, development of high or lowbone turnover and/or responsiveness to therapy for conditions relatingto bone density or bone turnover.
 2. A method as claimed in claim 1 inwhich the analysis comprises restriction fragment length polymorphismusing endonuclease digestion.
 3. A method as claimed in claim 2 in whicha segment of the vitamin D receptor is amplified using polymerase chainreaction prior to endonuclease digestion.
 4. A method as claimed inclaim 2 in which the endonuclease is selected from the group consistingof Bsm1, Apa1, EcoRV, Taq1, and isoschizomers thereof.
 5. A method asclaimed in claim 4 in which the restriction endonuclease is Bsm1.
 6. Amethod as claimed in any one of claims 3 to 5 in which the segment ofthe vitamin D receptor is amplified using a pair of primers selectedfrom the group consisting of5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' (SEQID:NO 2) and 5'-AACCAGCGGAAGAGGTCAAGGG-3' (SEQ ID:NO 3); and5'-CAGAGCATGGACAGGGAGCAAG-3' (SEQ ID:NO 4) and5'-GCAACTCCTCATGGCTGAGGTCTCA-3' (SEQ ID:NO 5).
 7. A method as claimed inclaim 1 in which the segment of the vitamin D receptor gene analysedrepresents a variableportion of the vitamin D receptor or gene regionsin linkage with at least one of the Bsm1, Apa1, EcoRV and Taq1 cutsites.